OKRATOKSİN A TAYİNİ İÇİN MONOLİTİK AFİNİTE KOLONLARININ HAZIRLANMASI VE İKİ-BOYUTLU HPLC SİSTEMİNDE ÇEVRİMİÇİ ANALİZLERİN İNCELENMESİ

Abstract

The aim of this thesis is the preparation of monolithic affinity columns and simultaneous determination of Ochratoxin A (OTA) in food by assessing tolerable levels for humans with two-dimensional HPLC (2D-HPLC) system. It was aimed to combine the hot topics of recent researches such as monolithic columns, multidimensional chromatographic techniques and mycotoxin analysis in foodstuffs. Hydrophobic affinity-based monolithic columns were designed in steel HPLC columns to create alternative columns for disposable OTA immunoaffinity columns on the market. First, the N-methacryloyl-L-phenylalanine (MAPA) monomer which is polymerizable derivative of the L-phenylalanine molecule used as the hydrophobic ligand, was synthesized. Monolithic columns were synthesized by the bulk polymerization of MAPA and 2-hydroxyethyl methacrylate (HEMA) conducted in a steel HPLC column at 75°C (temperature controlled water bath) for 3.5 h. Monolithic columns are characterized by scanning electron microscopy (SEM), elemental analysis, Fourier transform infrared-attenuated total reflectance spectroscopy (FTIR-ATR), Raman spectroscopy and back pressure-flow rate dynamics. In the first step, optimization of the analysis conditions for OTA with the monolithic affinity columns prepared was studied in a one-dimensional HPLC (1D-HPLC) system. In this context, OTA concentrations (0.5-20 ng/mL), MAPA monomer content (0-250 μmol), mobile phase [ACN:H2O (containing 2% HAc) (40:60-45:55-51:49-55:60)], flow rate (0.25-1.5 mL/min), temperature (25°C-40°C) and injection volume (10-100 μL) were investigated. In the second step of the study, two different columns in the first dimension hydrophobic based monolithic columns as separation column and in the second dimension reverse phase C18 column as analytical column were integrated into HPLC system. OTA in foodstuffs (beer, wine, corn and coffee) was simultaneously analyzed in a two-dimensional HPLC system. The intra-day RSD% were varied from 0.21%-0.49% with recoveries obtained varying between 104.44% and 107.33% whereas the inter-day RSD% were varied from 0.44%-1.31% with recoveries found to be 104.34%-107.03% to the concentration of OTA at 2-5 ng/mL. It has been demonstrated that monolithic columns could be used for OTA analysis to alternative single-use disposable immunoaffinity columns on the market, which can be used repeatedly without significant loss in intra-day and inter-day reusability values.